Multiple binding of repressed mRNAs by the P-body protein Rck/p54

Abstract

Translational repression is achieved by protein complexes that typically bind 39 UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 59 extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

“…Biophysical studies of these complexes have ascribed modulatory roles for mIF4G domains in regulating RNA-binding and release, ATPase activity, and helicase function of their respective DEAD-box protein partners (Weirich et al 2006;Schutz et al 2008;Montpetit et al 2011;Buchwald et al 2013). Thus far, Human DDX6 directly interacts with CNOT1 www.rnajournal.org 1405 DDX6 has been characterized as a weak ATPase with a high affinity for RNA (Dutta et al 2011;Ernoult-Lange et al 2012), and yeast Dhh1p does not detectably unwind RNA secondary structures in vitro (Dutta et al 2011). It is possible that the CNOT1 mIF4G domain is capable of stimulating a latent helicase activity within DDX6.…”

“…DDX6 belongs to the DEAD-box family of proteins, and plays an evolutionarily conserved role in translational repression and the activation of mRNA decapping (Coller et al 2001;Nakamura et al 2001;Fischer and Weis 2002;Minshall and Standart 2004;Coller and Parker 2005;Fenger-Gron et al 2005;Carroll et al 2011;Ernoult-Lange et al 2012). Mechanistic effects of the yeast DDX6 ortholog, Dhh1p, on gene expression have been well studied, with translation being repressed at the initiation phase in a nutrient-responsive manner (Coller and Parker 2005).…”

The role of disordered protein regions in the assembly of decapping complexes and RNP granules

“…Multivalent binding is further ensured by homotypic interactions mediated by oligomerization domains (e.g., DCP1 trimerization and EDC3 dimerization) and heterotypic interactions mediated by SLiM mutimerization (e.g., HLMs in DCP2). In addition, decapping factors associate with RNA, and DDX6 at least has been shown to multimerize on RNA (Ernoult-Lange et al 2012). These RNA molecules can be bridged through interactions mediated by EDC3 or DCP1 oligomerization domains, resulting in multivalent networks.…”

DEAD box RNA helicase functions in cancer

“…Can both promote and repress translation initiation: associates with P-bodies, ATP hydrolysis allows release from P-bodies and translation initation [32][33][34][35][36][37] 58,65 Developmental roles in many species [57][58][59][60][61][62] Overexpression in several cancers [53][54][55][56] Stimulation of cell cycle progression and proliferation [70][71][72][73][74] Cell cycle progression/proliferation Dhh1p important for recovery from DNA damageinduced G1/S cell cycle arrest in yeast DDX6 required for S-phase progression and cell viability in HeLa and LoVo cells, 73,74 and proliferation in xenograft models 74 (siRNA studies)…”